Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize structurally conserved molecules that are derived from and unique to pathogens, referred to as pathogen-associated molecular patterns. As such, TLRs function in the mammalian immune system as front-line sensors of pathogen-associated molecular patterns, detecting the presence of invading pathogens (Takeuchi and Akira 2010 Cell 140:805-820). TLR engagement in sentinel immune cells causes biosynthesis of selected cytokines (e.g., type I interferons), induction of co-stimulatory molecules, and increased antigen presentation capacity. These are important molecular mechanisms that activate innate and adaptive immune responses. Accordingly, agonists and antagonists of TLRs find use in modulating immune responses. TLR agonists are typically employed to stimulate immune responses, whereas TLR antagonists are typically employed to inhibit immune responses (Gosu et al 2012 Molecules 17:13503-13529).
The human genome contains 10 known TLRs, of these TLR3, TLR7, TLR8, and TLR9 sense nucleic acids and their degradation products. The distribution of TLR7, TLR8, and TLR9 is restricted to the endolysosomal compartments of cells and they are preferentially expressed in cells of the immune system. In the activated, dimeric receptor configuration TLR7 and TLR8 recognize single stranded RNA at one ligand binding site and the ribonucleoside degradation products guanosine and uridine, respectively, (as well as small molecule ligands with related structural motifs) at a second ligand binding site (Zhang et al 2016 Immunity 45:737-748; Tanji et al 2015 Nat Struct Mol Biol 22:109-115). Engagement of TLR7 in plasmacytoid dendritic cells leads to the induction of interferon-α/β, which plays essential functions in the control of the adaptive immune response (Bao and Liu 2013 Protein Cell 4:40-52). Engagement of TLR8 in myeloid dendritic cells, monocytes, and monocyte-derived dendritic cells induces a prominent pro-inflammatory cytokine profile, characterized by increased production of tumor necrosis factor-α, interleukin-12, and IL-18 (Eigenbrod et al 2015 J Immunol 195:1092-1099). Thus, virtually all major types of monocytic and dendritic cells can be activated by agonists of TLR7 and TLR8 to become highly effective antigen-presenting cells, thereby promoting an effective innate and adaptive immune response. Most antigen presenting cell types express only one of these two receptors, accordingly small molecules with potent agonist bioactivity against both TLR7 and TLR8 receptors are potentially more effective immune adjuvants than agonists specific for only one of these TLRs. Thus a TLR7/TLR8 (TLR7/8) small molecule agonist with balanced, dual bioactivity would cause innate immune responses in a wider range of antigen presenting cells and other key immune cell types, including plasmacytoid and myeloid dendritic cells, monocytes, and B cells (van Haren et al 2016 J Immunol 197:4413-4424; Ganapathi et al 2015 PLoS One 10:e0134640). Such potent dual TLR7/8 agonists may also be effective in stimulating effective anti-tumor immune responses in cancer (Singh et al 2014 J Immunol 193:4722-4731; Sabado et al 2015 Cancer Immunol Res 3:278-287; Spinetti et al 2016 Oncoimmunol 5:e1230578; Patil et al 2016 Mini Rev Med Chem 16:309-322).
A number of small molecule structural classes are known to interact at the guanosine/uridine ligand binding site and possess varying levels of TLR 7 and/or TLR8 agonist bioactivity (see e.g., Lu et al 2012 Clin Cancer Res 18:499-509; U.S. Pat. Nos. 5,446,153, 6,194,425, 6,110,929, and 7,199,131), including derivatives of 1H-imidazo[4,5-c]quinoline that are TLR7 agonists or dual TLR7/8 agonists (see e.g., Vasilakos and Tomai 2013 Expert Rev Vaccines 12:809-819; U.S. Pat. No. 4,689,338). One such 1H-imidazo[4,5-c]quinoline is 1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine (Imiquimod), a TLR7-specific agonist that was approved in 1997 for the treatment of actinic keratosis, superficial basal cell carcinoma, and genital warts, and was subsequently approved for the treatment of basal cell carcinoma (see e.g., Hemmi et al 2002 Nat Immunol 3:196-200). While some 1H-imidazo[4,5-c]quinolines display selective TLR7 or TLR8 agonist activities, others display dual TLR7/8 agonist activities. For example, 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine was found to be a TLR7 agonist with negligible bioactivity against TLR8 (Shukla et al 2010 J Med Chem 53:4450-4465). In contrast, 2-propyl[1,3]thiazolo[4,5-c]quinolin-4-amine was found to be a TLR8 agonist with negligible activity against TLR7 (Gorden et al 2005 J Immunol 174:1259-1268). 1-(4-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (IMDQ) and 1-(3-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (meta-IMDQ) were found to be dual TLR7/8 agonists with potent agonist activity against both receptors (see e.g., Shukla et al 2010 J Med Chem 53:4450-4465; Shukla et al 2010 Bioorg Med Chem Lett 10:6384-6386; U.S. Pat. Nos. 8,728,486; 9,441,005).
However, rapid systemic distribution of soluble 1H-imidazo[4,5-c]quinoline-based TLR7/8 agonists following subcutaneous, intratumoral or intramuscular administration has been demonstrated to cause significant toxicities in patients (see e.g., Vasilakos et al 2013 Expert Rev Vaccines 12:809-819; Savage et al 1996 Br J Cancer 74:1482-1486; Pockros et al 2007 J Hepatol 47:174-182). Systemic immune system activation due to activation of TLRs in cells of the spleen and liver causes an increase in serum pro-inflammatory cytokine levels, which in turn causes flu-like symptoms and other adverse events that limits the utility of these compounds as human therapeutics to a topical route of administration. Thus, there remains a need for small molecule therapeutic agents with potent and balanced TLR7/8 agonist activities that also possess physiochemical properties that enable pharmaceutical compositions that promote retention of the compound at the site of injection.